A full-length R-like basic-helix-loop-helix transcription factor is required for anthocyanin upregulation whereas the N-terminal region regulates epidermal hair formation.

Earlier studies have shown that the Lc gene of maize, a member of the R gene family that encode basic-helix-loop-helix (bHLH) transcription factors, is involved with anthocyanin production and trichome formation in Arabidopsis. We previously reported that the N-terminus of R protein interacts with CAPRICE (CPC), a regulatory protein, in triggering epidermal hair differentiation in Arabidopsis. In this study, we investigated the roles of full-length R, the N-terminal region of R (RN) and the C-terminal region of R (RC) in epidermal cell differentiation and anthocyanin production. We found that the N-terminal region was responsible for leaf trichome and root hair differentiation, whereas full-length R was required for anthocyanin upregulation. Yeast two-hybrid analysis showed that the C-terminal region was the binding site for the formation of homo- or hetero-dimers of the R-like bHLH transcription factor. To stimulate anthocyanin production, full-length R is required.

Analysis of IIId, IIIe and IVa group basic-helix-loop-helix proteins expressed in Arabidopsis root epidermis.

Differentiation of Arabidopsis epidermal cells into root hairs and trichomes is a functional model system for understanding plant cell development. Previous studies showed that one of the Arabidopsis basic-helix-loop-helix (AtbHLH) proteins, GLABRA3 (GL3), is involved in root-hair and trichome differentiation. We analyzed 11 additional AtbHLH genes with homology to GL3. Estimation of the phylogeny based on amino acid sequences of the bHLH region suggests that 11 AtbHLH genes used in this study evolved by duplications of a single common GL3 ancestor. Promoter-GUS analysis showed that AtbHLH006, AtbHLH013, AtbHLH017 and AtbHLH020 were expressed in roots. Among them, AtbHLH006 and AtbHLH020 were preferentially expressed in root epidermal non-hair cells. Consistent with the expression patterns from promoter-GUS analysis, GFP fluorescence was observed in the nuclei of root epidermal non-hair cells of AtbHLH006p::AtbHLH006:GFP and AtbHLH020p::AtbHLH020:GFP transgenic plants. However, AtbHLH006 and AtbHLH0020 proteins did not interact with epidermis-specific MYB proteins and TTG1. Taken together, AtbHLH006 and AtbHLH020 may function in root epidermal cells, but other GL3-like bHLH proteins may have evolved to regulate different processes.

The Selaginella genome identifies genetic changes associated with the evolution of vascular plants.

Vascular plants appeared ~410 million years ago, then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes. We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first nonseed vascular plant genome reported. By comparing gene content in evolutionarily diverse taxa, we found that the transition from a gametophyte- to a sporophyte-dominated life cycle required far fewer new genes than the transition from a nonseed vascular to a flowering plant, whereas secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in posttranscriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the trans-acting small interfering RNA pathway, and extensive RNA editing of organellar genes.

Turnover of LeACS2, a wound-inducible 1-aminocyclopropane-1-carboxylic acid synthase in tomato, is regulated by phosphorylation/dephosphorylation.

1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is the rate-limiting enzyme of the ethylene biosynthesis pathway. ACS is regulated both transcriptionally and post-translationally. We previously reported that LeACS2, a wound-inducible ACS in tomato (Solanum lycopersicum), is phosphorylated in vivo, and suggested that phosphorylation regulates protein stability rather than enzymatic activity. In this report, we demonstrate that phosphorylation/dephosphorylation of LeACS2 regulates its turnover upstream of the ubiquitin-26S-proteasome degradation pathway. Pulse-chase experiments coupled with treatment with protein kinase/phosphatase inhibitors demonstrated that LeACS2 is stabilized by phosphorylation and degraded after dephosphorylation. The amount of LeACS2 affected by the protein kinase/phosphatase inhibitors significantly influenced cellular ACS activity, ACC content, and ethylene production levels in tomato fruit tissue, suggesting that post-translational regulation by phosphorylation plays an important role in the control of ethylene production. A calcium-dependent protein kinase (CDPK), LeCDPK2, was isolated as one of the protein kinases that are able to phosphorylate LeACS2 at Ser-460. LeACS2 was immediately phosphorylated after translation by CDPK and mitogen-activated protein kinase at different sites in response to wound signaling and almost all functional LeACS2 molecules are phosphorylated in the cell. Phosphorylation at both sites was required for LeACS2 stability.

The GLABRA2 homeodomain protein directly regulates CESA5 and XTH17 gene expression in Arabidopsis roots.

Arabidopsis root hair formation is determined by the patterning genes CAPRICE (CPC), GLABRA3 (GL3), WEREWOLF (WER) and GLABRA2 (GL2), but little is known about the later changes in cell wall material during root hair formation. A combined Fourier-transform infrared microspectroscopy-principal components analysis (FTIR-PCA) method was used to detect subtle differences in the cell wall material between wild-type and root hair mutants in Arabidopsis. Among several root hair mutants, only the gl2 mutation affected root cell wall polysaccharides. Five of the 10 genes encoding cellulose synthase (CESA1-10) and 4 of 33 xyloglucan endotransglucosylase (XTH1-33) genes in Arabidopsis are expressed in the root, but only CESA5 and XTH17 were affected by the gl2 mutation. The L1-box sequence located in the promoter region of these genes was recognized by the GL2 protein. These results indicate that GL2 directly regulates cell wall-related gene expression during root development.

Arabidopsis CAPRICE-LIKE MYB 3 (CPL3) controls endoreduplication and flowering development in addition to trichome and root hair formation.

CAPRICE (CPC) encodes a small protein with an R3 MYB motif and promotes root hair cell differentiation in Arabidopsis thaliana. Three additional CPC-like MYB genes, TRY (TRIPTYCHON), ETC1 (ENHANCER OF TRY AND CPC 1) and ETC2 (ENHANCER OF TRY AND CPC 2) act in a redundant manner with CPC in trichome and root hair patterning. In this study, we identified an additional homolog, CPC-LIKE MYB 3 (CPL3), which has high sequence similarity to CPC, TRY, ETC1 and ETC2. Overexpression of CPL3 results in the suppression of trichomes and overproduction of root hairs, as has been observed for CPC, TRY, ETC1 and ETC2. Morphological studies with double, triple and quadruple homolog mutants indicate that the CPL3 gene cooperatively regulates epidermal cell differentiation with other CPC homologs. Promoter-GUS analyses indicate that CPL3 is specifically expressed in leaf epidermal cells, including stomate guard cells. Notably, the CPL3 gene has pleiotropic effects on flowering development, epidermal cell size and trichome branching through the regulation of endoreduplication.

Functional analysis of the epidermal-specific MYB genes CAPRICE and WEREWOLF in Arabidopsis.

Epidermis cell differentiation in Arabidopsis thaliana is a model system for understanding the developmental end state of plant cells. Two types of MYB transcription factors, R2R3-MYB and R3-MYB, are involved in cell fate determination. To examine the molecular basis of this process, we analyzed the functional relationship of the R2R3-type MYB gene WEREWOLF (WER) and the R3-type MYB gene CAPRICE (CPC). Chimeric constructs made from the R3 MYB regions of WER and CPC used in reciprocal complementation experiments showed that the CPC R3 region cannot functionally substitute for the WER R3 region in the differentiation of hairless cells. However, WER R3 can substantially substitute for CPC R3. There are no differences in yeast interaction assays of WER or WER chimera proteins with GLABRA3 (GL3) or ENHANCER OF GLABRA3 (EGL3). CPC and CPC chimera proteins also have similar activity in preventing GL3 WER and EGL3 WER interactions. Furthermore, we showed by gel mobility shift assays that WER chimera proteins do not bind to the GL2 promoter region. However, a CPC chimera protein, which harbors the WER R3 motif, still binds to the GL2 promoter region.